Methods and kits for nucleic acid sequencing

ABSTRACT

Various embodiments of the present disclosure generally relate to molecular biological protocols, equipment and reagents for the sequencing of target nucleic acid (DNA, RNA, cDNA, etc) molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 13/062,208, filed Mar. 3, 2011 which is the national phase under 35U.S.C. § 371 of prior PCT International Application No.PCT/IB2009/006976 which has an International filing date of Sep. 3,2009, designating the United States of America, which claims the benefitof U.S. Provisional Patent Application Nos. 61/094,006 and 61/094,025filed on Sep. 3, 2008, the disclosures of which are hereby expresslyincorporated by reference in their entirety and are hereby expresslymade a portion of this application.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as fileSequenceListing_QMDX.001C1.TXT created and last modified on Oct. 17,2016, which is 622 bytes in size. The information in the electronicformat of the Sequence Listing is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to molecular biological methods, equipmentand reagents for sequencing target nucleic acid (DNA, RNA, cDNA, etc)molecules to enable both highly parallel and long ‘read-length’nucleotide sequencing.

Description of the Related Art

DNA is a long polymer consisting of units called nucleotides. The DNApolymers are long chains of single units, which together form moleculescalled nucleic acids. Nucleotides can be one of four subunits (adenine(A), cytosine (C), guanine (G) & thymine (T)) and, when in a polymer,they may carry the genetic information in the cell. DNA comprises twolong chains of nucleotides comprising the four different nucleotidesbases (e.g. AGTCATCGTAGCT (SEQ ID NO: 1) . . . etc) with a backbone ofsugars and phosphate groups joined by ester bonds, twisted into a doublehelix and joined by hydrogen bonds between the complementary nucleotides(A hydrogen bonds to T and C to G in the opposite strand). The sequenceof nucleotide bases along the backbone may determine individualhereditary characteristics.

The central dogma of molecular biology generally describes the normalflow of biological information: DNA can be replicated to DNA, thegenetic information in DNA can be ‘transcribed’ into mRNA, and proteinscan be translated from the information in mRNA, in a process calledtranslation, in which protein subunits (amino acids) are brought closeenough to bond, in order (as dictated by the sequence of the mRNA &therefore the DNA) by the binding of tRNA (each tRNA carries a specificamino acid dependant on its sequence) to the mRNA.

SUMMARY OF THE INVENTION

A method of sequencing a target polynucleotide is disclosed inaccordance with embodiments of the present invention. The methodcomprises: providing within an assay region a sensitive detectionnanostructure that generates a signal related to a property of thenanostructure within the assay region, wherein the nanostructure iscoupled to a means for detecting the signal; hybridizing within theassay region a primer to the 5′ end region of the target polynucleotide,such that the resulting primer-target polynucleotide is operably coupledto the nanostructure; adding one or more nucleotides and a polymerase tothe primer-target polynucleotide within the assay region underconditions that support polymerization of a nascent chain when at leastone of the added nucleotides is complimentary to the base on the targetpolynucleotide downstream of the primer; and detecting within the assayregion a change in the signal that is characteristic of the at least onenucleotide added to the nascent chain.

In preferred embodiments of the method, the property of thenanostructure is an electrical charge.

In certain embodiments of the method, the added nucleotide furthercomprises a charge mass reporter moiety comprising a high charge massmoiety and a linker. In some embodiments, the charge mass reportermoiety is configured to be removable. In some embodiments, the chargemass reporter moiety is removed from the added nucleotide afterdetecting the signal. In some other embodiments, the charge massreporter moiety is configured not to affect polymerization of thenascent chain by the polymerase. In yet other embodiments, the chargemass reporter moiety is configured to protrude out from the nascentchain so as to reach-down to the sensitive detection nanostructure.

In some embodiments, the high charge mass moiety comprises an aromaticand/or aliphatic skeleton comprising one or more of a tertiary aminogroup, an alcohol hydroxyl group, a phenolic hydroxy group, or anycombinations thereof. The high charge mass moiety may comprise one ormore of the following groups or derivatives thereof:

In certain embodiments of the method, the linker comprises a molecule ofthe following general formula:H₂N-L-NH₂wherein L comprises a linear or branched chain comprising an alkylgroup, an oxy alkyl group, or a combination thereof.

In some embodiments, L may comprise a linear chain comprising an alkylgroup, an oxy alkyl group, or a combination thereof. The number ofcarbon atoms in the linear chain may be 1 to 100.

In some embodiments, the added nucleotide may also comprise a cleavablecap molecule at the 5′ phosphate so that addition of another nucleotideis prevented until the cleavable cap is removed. In some otherembodiments, the linker can be bound to the 5′ phosphate group of theadded nucleotide, thereby acting as a cap.

In variations to the method, more than one nucleotide may be added tothe assay region, but a successive nucleotide is not added to thenascent chain until after the signal that is characteristic of thepreceding nucleotide added to the nascent chain is detected.

In some embodiments, the operable coupling between the primer-targetpolynucleotide and the nanostructure comprises immobilization of theprimer to the sensitive detection nanostructure. In other embodiments,the operable coupling between the primer-target polynucleotide and thenanostructure comprises immobilization of the target polynucleotide tothe sensitive detection nanostructure.

In certain embodiments, hybridizing the primer to the 5′ end region ofthe target polynucleotide comprises hybridizing the primer to anoligonucleotide that has been ligated to the 5′ end of the targetpolynucleotide.

In certain embodiments, the sensitive detection nanostructure isselected from the group consisting of a nanowire, a nanotube, a nanogap,a nanobead, a nanopore, a field effect transistor (FET)-type biosensor,a planar field effect transistor, and any conducting nanostructures.

The target polynucleotide and the primer preferably comprise moleculesselected from the group consisting of DNA, RNA, peptide nucleic acid(PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA),threose nucleic acid (TNA), synthetic nucleotide polymer, andderivatives thereof. The added nucleotide preferably comprises amolecule selected from the group consisting of a deoxyribonucleotide, aribonucleotide, a peptide nucleotide, a morpholino, a locked nucleotide,a glycol nucleotide, a threose nucleotide, a synthetic nucleotide, andderivatives thereof.

In some embodiments, the means for detecting the signal are selectedfrom the group consisting of piezoelectric detection, electrochemicaldetection, electromagnetic detection, photodetection, mechanicaldetection, acoustic detection and gravimetric detection.

An apparatus for sequencing a target polynucleotide is disclosed inaccordance with other embodiments of the present invention. Theapparatus comprises: an assay region comprising a sensitive detectionnanostructure capable of generating a signal related to the electricalcharge of the nanostructure, and a signal detection means coupled to thesensitive detection nanostructure. In some embodiments, the apparatusmay further comprise a pico-well or a microfluidics channel, arrayedwith the sensitive detection nanostructures, wherein the biologicalsample comprises any body fluid, cells and their extract, tissues andtheir extract, and any other biological sample comprising nucleotides.In some other embodiments, the apparatus may comprise a microfluidicscassette. The microfluidics cassette may comprise a sample receptionelement for introducing a biological sample comprising the targetpolynucleotide into the cassette; a lysis chamber for disrupting thebiological sample to release a soluble fraction comprising nucleic acidsand other molecules; a nucleic acid separation chamber for separatingthe nucleic acids from the other molecules in the soluble fraction; anamplification chamber for amplifying the target polynucleotide; an assayregion comprising an array of one or more sensitive detectionnanostructures that generate a signal related to a property of thenanostructures, wherein the assay region is configured to allow operablecoupling of the target polynucleotide to the nanostructures; and aconducting element for conducting the signal to a detector. In someexamples, the apparatus can be used for the biological sample, which canbe any body fluid, cells and their extract, tissues and their extract,and any other biological sample comprising the target polynucleotide.The apparatus for sequencing disclosed in some embodiments herein can beis sized and configured to be handheld.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and B are depictions of an illustrative embodiment ofimmobilization of a probe sequence and a measurement of a target DNA.

FIG. 2 is a depiction of an illustrative embodiment of some part of thesequencing by synthesis reaction.

FIG. 3 is a depiction of an illustrative embodiment of the addition of asynthetic nucleotide base to the nascent chain.

FIG. 4 provides an illustration of some embodiments of the cleavage ofthe linker and high charge mass reporter moiety ligated to thenucleotide.

FIG. 5 illustrates a depiction of the possible results from themeasurements from the sensitive detection nanostructures in someembodiments.

FIG. 6 illustrates an example of a polymerase complex.

FIG. 7 illustrates an example of microfluidics cassette designs forhandheld sequencing in some embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term nucleotide sequencing generally encompasses biochemical methodsfor determining the order of the nucleotide bases, adenine, guanine,cytosine, and thymine, in DNA or RNA molecules. The sequence of DNAconstitutes the heritable genetic information in genomes, plasmids,mitochondria, and chloroplasts that forms the basis for thedevelopmental programs of living organisms. Genetic variations can causedisease, or confer an increased risk of disease (although it is alsotrue that certain genetic variations confer beneficial traits). Thesevariations can be inherited (passed on by parents) or acquired(developed as an adult). It is therefore of significant importance toknow the sequence of these genetic molecules to gain a betterunderstanding of life, molecular systems and disease.

The advent of DNA sequencing has significantly accelerated biologicalresearch and discovery. The rapid speed of sequencing attainable withmodern DNA sequencing technology has been instrumental in thelarge-scale sequencing of the human genome, in the Human Genome Project.Related projects have generated the complete DNA sequences of manyanimal, plant, viral, and microbial genomes.

RNA sequencing, which for technical reasons is easier to perform thanDNA sequencing, was one of the earliest forms of nucleotide sequencing.The major landmark of RNA sequencing, dating from the pre-recombinantDNA era, is the sequence of the first complete gene and then thecomplete genome of Bacteriophage MS2, identified and published by WalterFiers and his coworkers at the University of Ghent (Ghent, Belgium),published between 1972 and 1976.

The chain-termination method developed by Frederick Sanger andco-workers in 1975 was the first method of DNA sequencing to be employedon a large scale. Prior to the development of rapid DNA sequencingmethods in the early 1970s by Sanger in England and Walter Gilbert andAllan Maxam at Harvard, a number of laborious methods were used. Forinstance, in 1973 Gilbert and Maxam reported the sequence of 24base-pairs using a method known as wandering-spot analysis.

In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencingmethod based on chemical modification of DNA and subsequent cleavage atspecific bases. The method requires radioactive labelling at one end ofthe DNA strand and purification of the DNA fragment to be sequenced. Infrequent breaks are generated at one and sometimes two of the fournucleotide bases and this repeated in four reactions (G, A+G, C, C+T).This produces a series of labelled fragments, from the radiolabelled endto the first ‘cut’ site in each molecule and size-separated by gelelectrophoresis, with the four reactions arranged side by side.Maxam-Gilbert sequencing was not readily taken up due to its technicalcomplexity, extensive use of hazardous chemicals, and difficulties withscale-up. In addition, the method cannot easily be customized for use ina standard molecular biology kit.

The chain-termination or Sanger method requires a single-stranded DNAtemplate, a DNA primer, a DNA polymerase, radioactively or fluorescentlylabeled nucleotides, and modified nucleotides, dideoxynucleotidestriphosphates (ddNTPs) that terminate DNA strand elongation. The DNAsample is divided into four separate sequencing reactions, containingthe four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and theDNA polymerase. To each reaction is added only one of the fourdideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). Thesedideoxynucleotides are the chain-terminating nucleotides, lacking a3′-OH group required for the formation of a phosphodiester bond betweentwo nucleotides during DNA strand elongation. Incorporation of adideoxynucleotide into the nascent (elongating) DNA strand thereforeterminates DNA strand extension, resulting in various DNA fragments ofvarying length. The dideoxynucleotides are added at lower concentrationthan the standard deoxynucleotides to allow strand elongation sufficientfor sequence analysis.

The newly synthesized and labeled DNA fragments are heat denatured, andseparated by size (with a resolution of just one nucleotide) by gelelectrophoresis on a denaturing polyacrylamide-urea gel. Each of thefour DNA synthesis reactions is run in one of four individual lanes(lanes A, T, G, C); the DNA bands are then visualized by autoradiographyor UV light, and the DNA sequence can be directly read off the X-rayfilm or gel image. In the image on the right, X-ray film was exposed tothe gel, and the dark bands correspond to DNA fragments of differentlengths. A dark band in a lane indicates a DNA fragment that is theresult of chain termination after incorporation of a dideoxynucleotide(ddATP, ddGTP, ddCTP, or ddTTP). The terminal nucleotide base can beidentified according to which dideoxynucleotide was added in thereaction giving that band. The relative positions of the different bandsamong the four lanes are then used to read (from bottom to top) the DNAsequence as indicated.

DNA fragments can be labeled by using a radioactive or fluorescent tagon the primer, in the new DNA strand with a labeled dNTP, or with alabeled ddNTP. There are some technical variations of chain-terminationsequencing. In one method, the DNA fragments are tagged with nucleotidescontaining radioactive phosphorus for radiolabeling. Alternatively, aprimer labeled at the 5′ end with a fluorescent dye is used for thetagging. Four separate reactions are still required, but DNA fragmentswith dye labels can be read using an optical system, facilitating fasterand more economical analysis and automation. This approach is known as‘dye-primer sequencing’. The later development by L Hood and co-workersof fluorescently labeled ddNTPs and primers set the stage for automated,high-throughput DNA sequencing.

The different chain-termination methods have greatly simplified theamount of work and planning needed for DNA sequencing. For example, thechain-termination-based “Sequenase” kit from USB Biochemicals containsmost of the reagents needed for sequencing, prealiquoted and ready touse. Some sequencing problems can occur with the Sanger method, such asnon-specific binding of the primer to the DNA, affecting accurateread-out of the DNA sequence. In addition, secondary structures withinthe DNA template, or contaminating RNA randomly priming at the DNAtemplate can also affect the fidelity of the obtained sequence. Othercontaminants affecting the reaction may consist of extraneous DNA orinhibitors of the DNA polymerase.

An alternative to primer labelling is labelling of the chainterminators, a method commonly called ‘dye-terminator sequencing’. Oneof major advantages of this method is that the sequencing can beperformed in a single reaction, rather than four reactions as in thelabeled-primer method. In dye-terminator sequencing, each of the fourdideoxynucleotide chain terminators is labeled with a differentfluorescent dye, each fluorescing at a different wavelength. This methodis attractive because of its greater expediency and speed and is now themainstay in automated sequencing with computer-controlled sequenceanalyzers (see below). Its potential limitations include dye effects dueto differences in the incorporation of the dye-labelled chainterminators into the DNA fragment, resulting in unequal peak heights andshapes in the electronic DNA sequence trace chromatogram after capillaryelectrophoresis. The dye-terminator sequencing method, along withautomated high-throughput DNA sequence analyzers, is now being used forthe vast majority of sequencing projects, as it is both easier toperform and lower in cost than most previous sequencing methods.

Modern dye-terminator or chain-termination sequencing can produce asequence that may have poor quality in the first 15-40 bases, a highquality region of 700-900 bases, and then quickly deteriorating quality.Automated DNA sequencing instruments (DNA sequencers) operating thesemethods can sequence up to 384 fluorescently labelled samples in asingle batch (run) and perform as many as 24 runs a day. However,automated DNA sequencers may carry out only DNA-size-based separation(by capillary electrophoresis), detection and recording of dyefluorescence, and data output as fluorescent peak trace chromatograms.Sequencing reactions by thermocycling, clean-up and re-suspension in abuffer solution before loading onto the sequencer may be performedseparately.

Recent so called NextGen sequencing technologies, are based onpyrosequencing these new high-throughput methods use methods thatparallelize the sequencing process, producing thousands or millions ofsequences at once.

As molecular detection methods are often not sensitive enough for singlemolecule sequencing, Helicos method may be an exception, most approachesuse an in vitro cloning step to generate many copies of each individualmolecule. Emulsion PCR is one method, isolating individual DNA moleculesalong with primer-coated beads in aqueous bubbles within an oil phase. Apolymerase chain reaction (PCR) then coats each bead with clonal copiesof the isolated library molecule and these beads are subsequentlyimmobilized for later sequencing. Emulsion PCR is used in the methodspublished by Marguilis et al. (commercialized by 454 Life Sciences,acquired by Roche), Shendure and Porreca et al. (also known as “polonysequencing”) and SOLiD sequencing, (developed by Agencourt and acquiredby Applied Biosystems). Another method for in vitro clonal amplificationis “bridge PCR”, where fragments are amplified upon primers attached toa solid surface, developed and used by Solexa (now owned by Illumina).These methods both produce many physically isolated locations which eachcontain many copies of a single fragment. The single-molecule methoddeveloped by Stephen Quake's laboratory (later commercialized byHelicos) skips this amplification step, directly fixing DNA molecules toa surface.

Once clonal DNA sequences are physically localized to separate positionson a surface, various sequencing approaches may be used to determine theDNA sequences of all locations, in parallel. “Sequencing by synthesis”,like the popular dye-termination electrophoretic sequencing, uses theprocess of DNA synthesis by DNA polymerase to identify the bases presentin the complementary DNA molecule. Reversible terminator methods (usedby Illumina and Helicos) use reversible versions of dye-terminators,adding one nucleotide at a time, detecting fluorescence corresponding tothat position, then removing the blocking group to allow thepolymerization of another nucleotide. Pyrosequencing (used by 454) alsouses DNA polymerization to add nucleotides, adding one type ofnucleotide at a time, then detecting and quantifying the number ofnucleotides added to a given location through the light emitted by therelease of attached pyrophosphates. “Sequencing by ligation” is anotherenzymatic method of sequencing, using a DNA ligase enzyme rather thanpolymerase to identify the target sequence. Used in the polony methodand in the SOLiD technology offered by Applied Biosystems, this methoduses a pool of all possible oligonucleotides of a fixed length, labeledaccording to the sequenced position. Oligonucleotides are annealed andligated; the preferential ligation by DNA ligase for matching sequencesresults in a signal corresponding to the complementary sequence at thatposition.

Other methods of DNA sequencing may have advantages in terms ofefficiency or accuracy. Like traditional dye-terminator sequencing, theyare limited to sequencing single isolated DNA fragments. “Sequencing byhybridization” is a non-enzymatic method that uses a DNA microarray. Inthis method, a single pool of unknown DNA can be fluorescently labeledand hybridized to an array of known sequences. If the unknown DNA canhybridize strongly to a given spot on the array, causing it to “lightup”, then that sequence is inferred to exist within the unknown DNAbeing sequenced. Mass spectrometry can also be used to sequence DNAmolecules; conventional chain-termination reactions produce DNAmolecules of different lengths and the length of these fragments canthen be determined by the mass differences between them (rather thanusing gel separation).

There are new proposals for DNA sequencing, which are in development,but remain to be proven. These include labeling the DNA polymerase(Visigen), reading the sequence as a DNA strand transits throughnanopores, or using nano-edge probe arrays that are stepped withsub-Angstrom resolution over a stretched and immobilized ssDNA (Reveo),a technique that uses single-photon detection, fluorescent labelling andDNA electrophoresis with detection using plasmonic nanostructures(base4innovation), and microscopy-based techniques, such as AFM orelectron microscopy that are used to identify the positions ofindividual nucleotides within long DNA fragments by nucleotide labelingwith heavier elements (e.g., halogens) for visual detection andrecording.

With exception of methods using mass spec, nanopores andmicroscopy-based techniques, several methods presently available, or indevelopment generally require the use of expensive optical equipment andcomplex software. Furthermore, mass spec, nanopores and microscopy-basedtechniques may require bulky equipment that may limit their deploymentand certainly can drive costs up. Various embodiments used in connectionwith the present disclosure look to perform long read length, highlyparallel, potentially single molecules sequencing in a portable and costeffect device using a novel sequencing by synthesis technique.

The sequencing of the human genome and the subsequent studies have sincedemonstrated the great value in knowing the sequence of a person's DNA.The information obtained by genomic DNA sequence analysis can provideinformation about an individual's relative risk of developing certaindiseases (such as breast cancer and the BRCA 1&2 genes). Furthermore,the analysis of DNA from tumors can provide information about stage andgrading.

Infectious diseases, such as those caused by viruses or bacteria alsocarry their genetic information in nucleotide polymer genomes (eitherDNA or RNA). Many of these have now been sequenced, (or enough of theirgenome sequenced to allow for a diagnostic test to be produced) and theanalysis of infectious disease genomes from clinical samples (a fieldcalled molecular diagnostics) has become one of important methods ofsensitively and specifically diagnosing disease.

Measurements of the presence or absence, as well as the abundance ofmRNA species in samples can provide information about the health statusof individuals, the disease stage, prognosis and pharmacogenetic andpharmacogenomic information. These expression arrays are fast becomingtools in the fight against complex disease and may gain in popularity asprices begin to fall.

In short, the analysis of nucleotide polymers (DNA & RNA) has becomeimportant in the clinical routine, however, cost remains a barrier towidespread global adoption. One reason for this is the complexity of theanalysis requiring expensive devices that are able to sensitivelymeasure up to four different fluorescence channels as RT-PCR experimentsprogress. The cheaper alternatives may require skilled technicians torun and interpret low-tech equipment, such as electrophoresis gels, butthis too may be expensive and a lack of skilled technicians indeveloping countries is prohibitive.

To solve this, a method of nucleotide polymer analysis that may requirecheap and easy to use devices may be required. Some embodiments of thepresent disclosure describes chemical reagents, synthetic nucleotides,that can generally be utilized in such devices. Various embodiments usedin connection with of the present disclosure describes novel syntheticnucleotides that comprises at least some standard nucleotides (or anymodifications, or isoforms), with a high negative charge mass reportermoiety attached via a linker molecule (for instance, attached to the 5′phosphate group), with the linker length of such a length so as toprotrude from a polymerase complex during polymerization, so as not tocause a significant deleterious effect on the polymerase's action.

The terms nucleic acid or oligonucleotide or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp. 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

As used in various embodiments herein, a nucleotide can be, but notlimited to, one of the following compounds, Adenine, Guanine, Cytosine,Thymine, Uracil, and Inosine as well as any modified nucleotides, anynucleotide derivatives and any degenerate base nucleotides.

Some non-limiting examples of such nucleotide may comprise adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof. Furthermore, single stranded deoxyribose nucleicacid (ssDNA) can generally be a single stranded nucleotide polymermolecule, comprising Nucleotides and double stranded deoxyribose nucleicacid (dsDNA) can generally be a double strand comprising two ssDNAmolecules linked together via, for example, hydrogen bonding, in areverse complimentary orientation.

Nucleotides can generally be synthesized through a variety of methodsboth in vitro and in vivo. This can involve salvage synthesis (there-use of parts of nucleotides in resynthesizing new nucleotides throughbreakdown and synthesis reactions in order to exchange useful parts), orthe use of protecting groups in a laboratory. In the latter case, apurified nucleoside or nucleobase can be protected to create aphosphoramidite, and can be used to obtain analogues not present innature and/or to create an oligonucleotide.

In some embodiments, nucleotide synthesis comprises the formation of anucleoside (the nitrogenous base joined to a sugar). The sugar involvedin the synthesis and structure of a nucleotide may be either ribose ordeoxyribose; in the latter case, the prefix ‘deoxy’ may be added beforethe name of the nucleoside in all cases except Uracil. A functionalgroup of phosphate can be then esterified to the sugar, creating anucleotide. The phosphate group may comprise one, two, or threephosphates, forming mono-phosphates, di-phosphates, or tri-phosphates,respectively.

Some other embodiments of the present disclosure describe the design,synthesis and use of special synthetic nucleotides comprising anucleotide and a reporter moiety, in which the reporter moiety may notact as a polymerase enzyme blocking moiety attached via a linker.

In various embodiments, the synthetic nucleotides can have at least someof the following aspects:

-   -   1. The reporter moieties report based upon charge mass, not        enzymatic activity, fluorescence etc so there can generally be        more flexibility;    -   2. Each synthetic nucleotide may carry a different charge        mass—although for simplicity initially the same charge mass can        be used for proof of principle;    -   3. The reporter moieties may be easily cleaved; and/or    -   4. The nucleotides may be cheaply and easily mass synthesized.

The one of possible positions available for the attachment of linkersand the reporter moieties, so as to not interfere with polymerization orhydrogen bonding between the bases of nucleotides when hybridizing withits compliment base in another nucleotide polymer (i.e. when two strandsof reverse compliment DNA hybridize to form a double stranded DNAmolecule), can be the phosphate linkage in the nucleotide.

Some other embodiments describe methods of the use in which the linkerand reporter moiety can be cleaved from the synthetic nucleotide in theiterative manner after detection; one of possible places to attach thelinker can be the 5′-phosphate end of phosphate linkage.

Nature of linkage with the 5′-phosphate: There are at least two optionsavailable which could facilitate synthesis at the 5′-phosphate terminal:

-   -   1. Thiophosphate; and/or    -   2. Phosphoramidate.

The proposed linker therefore can have the following structure at leastin some embodiments.H₂N-L-NH₂

Where, L could be, but is not limited to, any linear or branched chainmolecule that is configured to link to a nucleotide as well as a highcharge mass moiety, both of which are present in a synthetic nucleotide.In some embodiments, L comprises a plurality of an alkyl group, an oxyalkyl group or the combination thereof with various lengths. In oneembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 100. In another embodiment, the numberof an alkyl group, an oxy alkyl group or the combination thereof in L is1 to 75. In still another embodiment, the number of an alkyl group, anoxy alkyl group or the combination thereof in L is 1 to 50. In stillembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 25. In some other embodiments, thenumber of an alkyl group, an oxy alkyl group and the combination thereofin L can be more than 100. While NH₂ is presented for the purpose ofillustration, this NH₂ can be substituted with any other function groupthat can be cross-linked to a nucleotide or its derivative as well as ahigh charge mass moiety, both of which are present in a syntheticnucleotide. Some illustrative examples that can be used instead of NH₂include, but not limited to, any alkyl group (e.g. CH₂₊₁, wherein nrepresents a positive integer number such as 1, 2, 3, and etc), anyalcohol group (e.g. C—H₂—OH, wherein n represents a positive integernumber such as 1, 2, 3, and etc), any carboxyl group (e.g. COOH), anyamide group (e.g. CONH), and any derivatives thereof. As the linkermolecules can vary in length and chemical structure in part to enablethe reporter moiety to extend out from a nucleotide polymerase (e.g. DNApolymerase, RNA polymerase and others) complex so that some aspects ofpolymerization may not be influenced entirely or partially.

The easy access to the linkers of various lengths can be considered as abenefit in a situation where the desired length of the linker may not beknown completely or partially. This may make the optimizationexperiments easy.

The linker with the nucleotide (say Adenosine as an illustrativeexample) therefore may have the following structure at least in someembodiments. While adenosine is presented in some examples below, thisadenosine can be substituted with any other natural or syntheticnucleotide, any modifications thereof and any derivatives thereof insome other embodiments.

In some embodiments, various lengths of linkers at this position mayhave the following structures (exemplified with the Adenosine):

1. Ethylenediamine (2 carbon bond length separation)

2. Pentanediamine (5 carbon bond length separation)

3. Length equivalent to 13 carbon bond length separation

Thus in some embodiments, the linkers thus selected can be:

1. Easily available;

2. Easy to link and cleave (please refer the probable protocols below);and/or

3. Not to interact with the polymerase and the polynucleic acid strandand/or not affect nucleotide polymerization and growth of a nascentnucleotide polymer.

The reporter moiety: In some embodiments, the reporter moieties can beassociated with the other properties like the chromophoric nature forenabling their detection by UV or visible detector or the fluorescentnature making them to be detected by the fluorimetric detection.

The charge on the reporter: certain embodiments of the present inventiondescribe the reporter moiety to carry a large charged mass. In oneembodiment, the reporter moiety may introduce a higher charge mass tothe synthetic nucleotide than the charge mass of the nucleotide or itsderivative, which is present in the synthetic nucleotide. However, inanother embodiment, the charge mass introduced by the reporter moietycan be substantially equal to or less than the charge mass of thenucleotide or its derivative, which is present in the syntheticnucleotide. Some non-limiting and illustrative examples of a reportermoiety are provided in this specification. These examples are providedonly for the illustration purpose and therefore should not be consideredto limit the scope of the invention. The chemical structure and/ordimension (e.g. length, size, and mass of a molecule used as a reportermoiety) of a reporter moiety may not be restricted as long as thereporter moiety is configured to provide a charge mass to the syntheticnucleotide and also not to affect polymerization reaction of nucleotidespartially or entirely.

The charge on the moiety can be positive or negative. Taking intoconsideration the nature of linkage, the following provides some aspectsof the selection of charge that can be possibly used in some embodimentsof the present disclosure. In preferred embodiments, the charge issufficient to cause a detectable change in a property (e.g., electricalresistance) of a sensitive detection nanostructure (e.g., a nanowire)which is operably coupled to the template sequence to which a syntheticnucleotide comprising the reporter moiety is added, e.g., duringsequencing by synthesis.

Positive charge: In some embodiments, the large number of positivecharges can generally be induced on the reporter moiety through theincorporation of tertiary amino groups on the aromatic or aliphaticskeleton. In such embodiments, in turn in the acidic pH (less than 7),these groups may acquire the positive charges making them detectable.

Negative charges: In some other embodiments, the negative charges cangenerally be induced on the reporter moiety through the incorporation ofalcohol hydroxyl and/or phenolic hydroxy functionalities on the aromaticor aliphatic skeleton. Given below are some of the proposed reportermoieties which meet the above mentioned criteria. The fragments listedbelow may be available and able to link to the linker through the aminoterminal. The additional advantage could be that the reagents that areproposed for the phosphoramidate linkage formation may be the same asthis amide linkage formation (Therefore reducing costs of the systemfurther).

Moreover, at least in part due to the stability of this linkage to thealkaline pH (above 7), the process of induction of negative charge wouldbe of no or substantially small interference. For the purpose ofillustration, the following three non-limiting examples are presented.These examples are provided only for the purpose of illustration andtherefore should not be considered to limit the scope of the invention.As such, any modifications on the following examples are certainlyincluded in the scope of the invention. For example, any substitution ofone or more groups (e.g. —OH, ═O, COOH, and others) linked to theexamples can be practiced. Also oligomerization or polymerization of oneof more of the following examples can also be permitted. Further anyother chemical structure or molecule with various dimensions (e.g.length, size, and mass of the reporter moiety) can be used as a reportermoiety if such chemical structure or molecule is configured to provide acharged mass to the synthetic nucleotide and also not to affectpolymerization reaction of nucleotides partially or entirely.

After acquiring the charges, some of these reporters in certainembodiments may exist as follows,

Whereas, the reporter-1 and reporter-3 may be available on shelf,reporter-2 may be custom synthesized.

The reporter moieties proposed can generally (be) thus:

1. Easily available or synthesizable;

2. bear a large charge;

3. Not costly; and/or

4. easy to link and cleave.

Final compounds (monomers): Based on the above propositions, the finalstructures of the nucleotides along with the linkers and the reporterswould be as follows at least in certain parts of embodiments. Thefollowing examples of some final compounds are also provided for thepurpose of illustration and therefore should not be considered to limitthe scope of the invention. As described above, any variations permittedfor a nucleotide or its derivative, a linker and a charge mass reportermoiety are also permitted to a final compound. Thus, for the adenosineas a nucleotide at the 5′-phosphate terminal in some examples, if thelinker is, say, C13 equivalent (option 3 above), the various linkerswould make the final structures looks as below:

One proposed final synthetic nucleotide-1 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

Another proposed synthetic nucleotide-2 (note the reporter is in monomerform and this can be increased by aggregating these monomers to increasecharge mass as required):

Still another proposed synthetic nucleotide-3 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

The following is a non-limiting, illustrative example of synthesisprotocols used in at least some embodiments:

-   -   1. Synthesis of 5′-phosphoramidates of Adenosine: (Linkage of        Nucleotide with the diamine linker). Method of Chu et all can be        used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamantes and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and was        incubated at room temperature with constant stirring. The        reaction was monitored till completion.    -   2. Synthesis of Final proposed structures: (Linkage of the        diamine linker with reporter moiety). Method of Chu et all can        be used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamines and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and        then incubated at room temperature with constant stirring. The        reaction was monitored till completion.

One advantage of the similar procedure is that it may work out for boththe steps leading to the formation of final compounds as monomers.

In some illustrative examples of some embodiments, (see below) cleavageof the linkers and reporter moieties may need to be done. The linkageslike phosphoramidates can generally be rather readily cleaved by the useof acids like Trifluoroacetic acid at an ambient temperature.

By way of an illustrative example, the proposed synthetic nucleotide-2demonstrated as a probable 3D view below. The aromatic ring at thebottom left of the molecule bears three hydroxy functions which couldpotentially get converted to the negative charge under slight alkalineconditions. Following is the 3D conformation of the Adenosine attachedwith the Reporter −1 through linker 3 and the related data.

Approximate distance between the phosphoramidate and terminal chargedatom may be about 20 angstroms, which could generally be sufficient toinduce the charge potential in the surface for detection. This distancecan further be altered with the further modifications in the phase atleast in part by changing the linker lengths. The charge on the terminalreporter moieties can also be changed by the variations in the chemistryof reporter moieties.

One aspect of the present disclosure describes a novel sequencing bysynthesis technology. Sequencing by synthesis can be the general termused for determining the sequence of a single strand DNA molecule bygrowing the nascent, reverse compliment, strand and detecting theaddition of each new nucleotide in the growing polymer. Using the moremodern methods described above (methods employed by Helicos, 454 LifeSciences & Solexa), this can be performed by adding each separatenucleotide (adenine, guanine, cytosine or thymine) separately, in thepresence of a polymerase and other elements required for polymerization,with a fluorescent reporter moiety ligated to the nucleotide and thenobserving the fluorescence using sensitive optical detection equipment.If there is fluorescence in the correct spectra for that nucleotideaddition step, then the ‘base calling’ bioinformatic program may add theappropriate base in sequence. The reaction can then be washed and thenext nucleotide in the cycle (wherein each of the four nucleotidesAdenince, Guanine, cytoisine and Thyomine (or uracil for RNA) are addedsequenntially) can be added. This cycle can be repeated until betweenapproximately 25 bp to 900 bp or more (for example, depending on whichmethod is used) worth of sequence data is obtained for each reaction(Reports from the market suggest read lengths of up to 900 bp are nowpossible using Roche's (ex 454) genome sequencer). To enable wholegenome sequencing, many thousands of these reactions can be performed inparallel.

In some embodiments, the present sequencing methods and components candetect the addition of nucleotides by sensing their innate electricalcharge, or the charge of a ligated ‘high charge-mass’ reporter moiety,using microfluidic chips, or reaction ‘plates’ arrayed with sensitivenanostructures that are capable of detecting a differences in chargedensities at or near their surface (nanowire or nanotube FET biosensors,piezo-electric films, etc), or as molecules pass through them(nanopores), instead of using fluorescence and expensive opticaldetection equipment. Thus when a new nucleotide is added to the growingpolymer in a sequencing by synthesis reaction the charge at, or near thesurface (or through a nanopore) of the sensitive nanostructure mayincrease (due to the addition of the negative nucleotide and in someembodiments, the negative nucleotide and it's high charge-mass reportermoiety, being added) and this can be detected (for instance, if were touse a nanowire as the detecting structure, an increase in charge causedby the addition of a nucleotide close to its surface will be detected bya change in resistance in the wire, due to a phenomenon called the fieldeffect) by a change in property in the sensitive detectionnanostructure. However, as the polymer grows, the signal may diminish asthe charges carried by the nucleotides being added may be too far awayfrom the sensitive nanostructure (e.g. nanowire) to illicit a change inproperty of the sensitive detection nanostructure and no signal may beobserved. Therefore, the ‘read length’ (amount of sequence data that isable to be obtained by this method of nucleotide sequencing) may belimited.

Some embodiments of the present disclosure address this limitation, atleast in part by using synthetic nucleotides that comprise normalnucleotides, with a high negative charge mass reporter moiety attachedvia a linker molecule (for instance, attached to the 5′ phosphate group,or the base group), with the linker length increasing as the reactionprogresses. This high charge mass can generally be designed to ‘reachdown’ to the sensitive nanostructure (e.g. nanowire) to cause a changein property of the sensitive detection nanostructure (e.g. a fieldeffect or other piezo-electric change in the structure depending on thesensitive detection nanostructure used). To enable a good qualitycontrol measure and to ensure long read lengths by eliminating the buildup of many reporter moieties which would cause an ever increasing fieldeffect, these reporter moieties can be cleaved to allow for the additionof the next nucleotide in the sequencing by synthesis sequence. Thecleavage of the reporter moiety effectively ‘resets’ the system allowingfor clear signals and a more improved signal to noise ration than ifusing natural nucleotides without the linker plus high charge-massreporter moiety.

Therefore, in some embodiments, the cyclical reaction comprises at leastsome or all of the following series of events:

-   -   1. The template molecule (DNA, RNA, or other synthetic        nucleotide polymer molecule such as PNA or morpholino oligos) to        be sequenced can either be ligated to the sensitive detection        nanostructure and a primer added, bind to a pre-immobilized        primer sequence on the sensitive detection nanostructure, or        uncoiled and elongated in a microfluidics channel arrayed with        sensitive detection nanostructures;    -   2. The sensitive detection nanostructures can be washed with        water, or a low salt buffer (such as 1×SSC);    -   3. A measure of the sensitive detection nanostructure can be        made (baseline measurement);    -   4. A mixture containing one synthetic nucleotide, the polymerase        and other elements required for the polymerisation reaction can        be added. If the nucleotide added is complimentary to the base        on the template molecule immediately after the primer sequence,        it may be incorporated into the growing ‘nascent’ chain by the        polymerase;    -   5. The reaction can then be washed, for instance with either        water or a low salt buffer (such as, but not limited too,        1×SSC);    -   6. A measure of the sensitive detection nanostructure may be        made which may observe the effect caused by the high charge mass        of the reporter moiety (intermediate measurement);    -   7. The reporter moiety can then be cleaved (for instance by an        acid solution or enzymatically)    -   8. The reaction can then be washed, for instance with either        water or a low salt buffer (such as, but not limited to, 1×SSC);    -   9. A measurement of the sensitive detection nanostructure may be        made which may observe the effect of just the added nucleotide        without the linker and high charge mass reporter moiety        (Baseline+1 measurement); and/or    -   10. Points 2 through 9 may be repeated for each of the four        nucleotides. And this can be repeated repeatedly until a clear        signal degrades.

For some embodiments wherein the template molecule is immobilized to, orbound to a probe that can be in turn immobilized to the sensitivedetection nanostructure, the linker lengths that attach the high chargereporter moiety to the synthetic nucleotides may increase to enable thecharge to ‘reach down’ to the sensitive detection nanostructure to exertan effect. This may be necessary at least in some embodiments as thegrowing nucleotide polymer may move the next nucleotide addition sitefarther and farther from the sensitive detection nanostructure as thesequencing by synthesis reaction progresses. By increasing the length ofthe linker lengths as the reaction continues, the charge mass reportermoiety will still ‘reach down’ to the sensitive detection nanostructureto illicit change, even though the addition of the nucleotides isfurther from the sensitive detection nanostructure than would normallyallow a signal, as the nascent chain grows.

For some other embodiments wherein the template molecules is notimmobilized to the sensitive detection nanostructure, or hybridized to aprimer/probe that can be in turn immobilized to the sensitive detectionnanostructure, and can be instead free or immobilized horizontallyacross a cluster of sensitive detection nanostructures, a single linkerlength can be used for each of the cycle reactions.

Some aspects of embodiments of the present disclosure describe a methodof sequencing nucleotide polymers (DNA or RNA) by incorporatingsynthetic nucleotides, ligated, via a linker molecule to a reportermoiety, in a ‘sequencing by synthesis’ reaction. As used herein invarious embodiments, ‘sequencing by synthesis’ generally describes amethod of nucleotide sequencing wherein the addition of each nucleotideto the nascent chain (i.e. the growing nucleotide polymer, reversecompliment to the template nucleotide polymer) can be detected in realtime. Some other embodiments, rather than detecting nucleotide monomeradditions to the nascent chain by using the traditional opticaldetection of fluorescent labels (as used in presently availablesequencing by synthesis techniques), can detect each nucleotide additionbased upon the charge mass of the nucleotide monomer. In some otherembodiments, the charge mass of a covalently ligated charge massreporter moiety, using a sensitive detection nanostructure, or otherstructure capable of detection minute may change in surface charges.

As used herein in some aspects of embodiments, a “sensitive detectionnanostructure” can generally be any structure (nanoscale or not) capableof generating a signal in response to a change in a property of thenanostructure within an assay region. As used herein an “assay region”refers generally to the area or region in which the nanostructure ornanostructures at least partially reside, and in which thenanostructure(s) is operably coupled to a biomolecule (nucleic acid,primer, target sequence, etc.) e.g., via a direct or indirect bond orlinkage, covalent or not, or just in close enough physical proximity toexhibit a change in property and generate a signal in response to achange in the biomolecule. In preferred embodiments, such a change inproperty may be caused by a change in charge of a biological moleculeoperably coupled to the nanostructure within the assay region.Typically, the nanostructure is sensitive to changes at or near itssurface (such as with nanowire or carbon nanotube FET biosensors), or asmolecules pass through it (such as nanopore biosensors)—although theassay region may extend beyond the surface of the nanostructure toinclude the entire region within the field of sensitivity of thenanostructure. The nanostructure is preferably also coupled to adetector that is configured to measure the signal and provide an outputrelated to the measured signal. At any point along the length of thenanostructure, it may have at least one cross-sectional dimension lessthan about 500 nanometers, typically less than about 200 nanometers,more typically less than about 150 nanometers, still more typically lessthan about 100 nanometers, still more typically less than about 50nanometers, even more typically less than about 20 nanometers, stillmore typically less than about 10 nanometers, and even less than about 5nanometers. In other embodiments, at least one of the cross-sectionaldimensions can be less than about 2 nanometers, or about 1 nanometer. Inone set of embodiments the sensitive detection nanostructure can be atleast one cross-sectional dimension ranging from about 0.5 nanometers toabout 200 nanometers.

As used in various embodiments, a nanowire is an elongated nanoscalesemiconductor which, at any point along its length, has at least onecross-sectional dimension and, in some embodiments, two orthogonalcross-sectional dimensions less than 500 nanometers, preferably lessthan 200 nanometers, more preferably less than 150 nanometers, stillmore preferably less than 100 nanometers, even more preferably less than70, still more preferably less than 50 nanometers, even more preferablyless than 20 nanometers, still more preferably less than 10 nanometers,and even less than 5 nanometers. In other embodiments, thecross-sectional dimension can be less than 2 nanometers or 1 nanometer.In one set of embodiments the nanowire has at least one cross-sectionaldimension ranging from 0.5 nanometers to 200 nanometers. Where nanowiresare described having a core and an outer region, the above dimensionsrelate to those of the core. The cross-section of the elongatedsemiconductor may have any arbitrary shape, including, but not limitedto, circular, square, rectangular, elliptical and tubular. Regular andirregular shapes are included. A non-limiting list of examples ofmaterials from which nanowires of the invention can be made appearsbelow. Nanotubes are a class of nanowires that find use in the inventionand, in one embodiment, devices of the invention include wires of scalecommensurate with nanotubes. As used herein, a “nanotube” is a nanowirethat has a hollowed-out core, and includes those nanotubes know to thoseof ordinary skill in the art. A “non-nanotube nanowire” is any nanowirethat is not a nanotube. In one set of embodiments of the invention, anon-nanotube nanowire having an unmodified surface (not including anauxiliary reaction entity not inherent in the nanotube in theenvironment in which it is positioned) is used in any arrangement of theinvention described herein in which a nanowire or nanotube can be used.A “wire” refers to any material having a conductivity at least that of asemiconductor or metal. For example, the term “electrically conductive”or a “conductor” or an “electrical conductor” when used with referenceto a “conducting” wire or a nanowire refers to the ability of that wireto pass charge through itself. Preferred electrically conductivematerials have a resistivity lower than about 10⁻³, more preferablylower than about 10⁻⁴, and most preferably lower than about 10⁻⁶ or 10⁻⁷ohm-meters.

Nanopore generally has one or more small holes in an electricallyinsulating membrane. Nanopore is generally a spherical structure in ananoscale size with one or more pores therein. According to someaspects, a nanopore is made of carbon or any conducting material.

Nanobead is generally a spherical structure in a nanoscale size. Theshape of nanobead is generally spherical but can also be circular,square, rectangular, elliptical and tubular. Regular and irregularshapes are included. In some examples, the nanobead may have a poreinside.

Nanogap is generally used in a biosensor that consists of separationbetween two contacts in the nanometer range. It senses when a targetmolecule, or a number of target molecules hybridize or binds between thetwo contacts allowing for the electrical signal to be transmittedthrough the molecules.

The foregoing nanostructures, namely, nanowire, nanotube, nanopore,nanobead, and nanogap are described to provide the instant illustrationof some embodiments, and not for limiting the scope of the presentinvention. In addition to the foregoing examples, any nanostructure thathas a nanoscale size and is suitable to be applied to nucleic acidsequencing methods and apparatus as disclosed in the application shouldalso be considered to be included in the scope of the invention.

In general, nucleotide sequencing strategies for use with nanostructuresor nanosensors is to sense the charge at, or near the surfaces, oracross a nanogap or nanopore, which cause a measurable change in theirproperties (such as field effect transistors, nanogaps, or piezoelectricnanosensors). The charge sensed by the nanostructure can be originatedfrom the nucleotide complementary to the template nucleotide that isadded during the sequencing reaction. In some embodiments, thecomplementary nucleotide added during the sequencing reaction is linkedto a linker and a high charge mass reporter moiety, which are describedin detail elsewhere in the specification.

Field effect generally refers to an experimentally observable effectsymbolized by F (on reaction rates, etc.) of intramolecular coulombicinteraction between the centre of interest and a remote unipole ordipole, by direct action through space rather than through bonds. Themagnitude of the field effect (or ‘direct effect’) may depend on theunipolar charge/dipole moment, orientation of dipole, shortest distancebetween the centre of interest and the remote unipole or dipole, and onthe effective dielectric constant. This is exploited in transistors forcomputers and more recently in DNA field-effect transistors used asnanosensors.

Field-effect transistor (FET) is generally a field-effect transistor,which may use the field-effect due to the partial charges ofbiomolecules to function as a biosensor. The structure of FETs can besimilar to that of metal-oxide-semiconductor field-effect transistor(MOSFETs) with the exception of the gate structure which, in biosensorFETs, may be replaced by a layer of immobilized probe molecules whichact as surface receptors.

In some embodiments, the sequencing reaction may begin by hybridizing ashort ‘probe’ or ‘primer’ nucleic acid molecule, often referred to as anoligonucleotide, wherein a ‘probe’ or ‘primer’ can generally be a singlestranded nucleotide polymer molecule, ssDNA, RNA, PNA, Morpholino, orother synthetic nucleotide, to the 5′ end of the ‘target’ nucleotidemolecule, wherein the ‘target’ molecule can generally be the nucleotidepolymer of interest. It is the nucleotide sequence of this singlestranded nucleotide polymer molecule, ssDNA or RNA that can be to bedetermined. Furthermore, the ‘probe’ or ‘primer’ sequence can generallybe reverse complimentary to the ‘target’ nucleic acid molecule to besequenced and sufficiently long to facilitate hybridization-primerdesign is well known to those skilled in the art and many commercial andfreeware software platforms are available to facilitate primer, or probedesign.

In some embodiments, short adaptamers (another short oligonucleotide ofknown sequence) can generally be ligated to the target nucleotidepolymer and the primer or probe can be designed to be reverse complimentto this oligonucleotide sequence. In some embodiments the primer orprobe molecule can be immobilized on a sensitive detection nanostructureand the target nucleotide molecule can be hybridized to the primer. Inother embodiments, the target molecule can be first immobilized to thesensitive detection nanostructure and primers or probes can behybridized. In yet another embodiment, both the target nucleotidemolecule and the primer and probe molecules may be free and notimmobilized, but positioned close to the sensitive detectionnanostructures, and at least in some embodiments the hybridized targetand primer or probe molecules may be sufficiently close to exert ameasurable change in properties in the sensitive detectionnanostructure. This might be by positioning the molecules in amicrofluidics environment, or other undefined methodologies. Thefollowing is a non-limiting, illustrative example of certain embodimentsof the present disclosure. In one example, the first step in thetechnique can be to take a series of measurements (for instance ameasure of resistance in a nanowire or other field effect transistor) ofthe sensitive detection nanostructure in air, the presence of water, ora low salt buffer (such as 1×SSC). This can provide the base level forthe property of the sensitive detection nanostructure that can be to bemeasured in response to ongoing reaction (e.g. the electrical resistancein a nanowire). The sequencing by synthesis reactions can be initiatedby adding a single nucleotide species (along with a polymerase and otherchemicals required for polymerization), such as Adenine to the reaction.As all nucleotides may posses a natural charge, following the additionof the Adenine in the nascent chain (assuming the next nucleotide in thetarget nucleotide polymer is a Thymine) the charge at, or close to, asensitive detection nanostructure, created by this extra Adenine in thenascent chain, may cause a measurable change in properties in thesensitive detection nanostructure, which may be recorded, following awash step to eliminate background noise from the high ionic PCR reactionmix.

Therefore, in certain examples, if there is a Thymine (or Uracil ifsequencing RNA) nucleotide next in the template nucleotide, then onceAdenine can be added to the sequencing by synthesis reaction, it can beincorporated in the nascent chain, by the polymerase (which can also beadded as part of the reaction) and a signal detected, at least in partdue to the change in electrical charge at, or near the surface of thesensitive nanostructure, caused by the extra nucleotide in the nascentchain. However, should either Cytosine, Guanine, or Thymine been addedinstead, then no nucleotide would have been added to the nascent chainand no change in signal would have been observed from the sensitivenanostructure.

In certain other embodiments, each nucleotide can be added, one afterthe other, to the reaction mix, with polymerase and MgCl₂, H₂O andbuffer to enable polymerization of a complimentary nucleotide additionto the target sequence in the nascent chain. After each nucleotideaddition, the reaction can be washed and the next nucleotide added. Thiscycle can be repeated for all four nucleotides. Once all fournucleotides can be added, the cycle can be repeated until the desiredsequence may be elucidated, or the signal may deteriorate.

In various embodiments, the synthetic nucleotides may comprise one ormore of nucleotides, Adenine, Guanine, Cytosine & Thymine, plus isoformsof these bases (such as Inosine) with a reporter moiety attached, forinstance, via a linker to the 5′ phosphate group. If the sensitivedetection nanostructure may not be sensitive enough to detect changescause by single nucleotides, some embodiments of the present disclosuredescribes the use of synthetic nucleotides that comprise nucleotideswith a charge mass reporter molecule ligated to the nucleotide via along linker molecule, sufficiently long enough to ensure that thereporter may not have a significant deleterious effect on polymeraseactivity. The linker molecules can vary in length, and enable thereporter moiety to extend out from the DNA Polymerase complex so thatpolymerization (nucleotide addition) may not be prevented, or undulyhindered. Furthermore, as the sequencing by synthesis reactionprogresses, the nucleotides added to the nascent chain can get furtherand further from the sensitive detection nanostructure, which maydiminish the signal. To combat this and to therefore provide longer readlengths, the linker molecule lengths can be increased to enable thereporter charge mass moieties to ‘reach down’ to the sensitive detectionstructures to provide a clear signal.

In some embodiments, as each nucleotide can be added to the nascentchain, the reporter moieties can build up and would eventually overloadthe sensitive detection structures at least in part due to theiraggregating charges. To combat this, in certain illustrative examples,some embodiments may assume that the charges can be cleaved, whether byenzymatic, chemical, light, or other methods, after each nucleotide canbe added and the signal in the sensitive reporter moiety recorded. Oncethe linker and the reporter moiety can be cleaved and washed away,another measurement in the sensitive detection structure can be taken toserve as a quality control, or to ‘reset’ the system to improve thesignal to noise ration, before the next nucleotide can be added to thereaction.

Homopolymer stretches may have a problem for fluorescence basedsequencing technologies, as a dinucleotide stretch may provide twice theintensity of fluorescence and the trinucleotide may stretch three timesthe intensity of fluorescence, and so on. This may cause significantdifficulties in interpretation and base calling.

Much like the some fluorescence based sequencing technologies, variousembodiments used in connection with the present disclosure can generallydifferentiate between di-, tri-, etc homopolymer stretches by measuringthe intensity of the signal (this can be possible if the sensitivedetection nanostructure can be capable of quantitative orsemi-quantitative measurements, such as some nanowire or carbon nanotubeFET biosensors are).

Alternatively, if the sensitive detection nanostructure may not be ableto differentiate between dinucleotide, tri-nucleotide and otherhomopolymer additions, some embodiments may use synthetic nucleotidesdesigned to allow the addition of one nucleotide and therefore preventthe addition of other nucleotides. One illustrative method to achievethis can be to link the reporter moiety to the 5′ phosphate group, whichmay prevent further nucleotide additions to the nascent chain. Once thereporter and linker are cleaved, the next nucleotide can be added.Another illustrative method can be to place a cleavable cap molecule atthe 5′ phosphate to prevent further nucleotide additions to the nascentchain, with the linker and reporter moiety linked to another site on thenucleotide. Cap removal may then allow for the next nucleotide to beadded and the process repeated.

In some embodiments of this disclosure, all nucleotides can be added tothe reaction mix, for example, at the same time, however each of thesynthetic nucleotides may have a different distinct charge mass reportermoiety, at least in some embodiments. Measurements of the sensitivedetection nanostructures can be taken about every 2-3 ms, to mimic thespeed at which polymerase may add nucleotides to the nascent chain,which can be estimated at about 3 ms at a temperature of about 65° C. Insuch embodiment, the reporter moieties may not be cleaved, so the readlengths may be shorter at least in part due to the signal to noise ratiodecreasing as the nascent chain may extend and charge reporter moietiescan build up, but sequencing time can be much quicker. In certainembodiments, a synthetic polymerase, that can be engineered to catalyzethe polymerization reaction slower.

EXAMPLES

The followings are some illustrative and non-limiting examples of someembodiments of the present disclosure.

Example 1—DNA Sequencing

The sequencing methodology in one example may not use fluorescence andexpensive sensitive cameras, but instead may detect the addition of thesynthetic nucleotides described in some aspects of the presentdisclosure, at least in part by sensing the electrical charge ofreporter moiety, using sensitive nanostructures that may be capable ofdetecting a build up of charge mass within the assay region, e.g., at ornear the surface of the sensitive detection nanostructure. When a newnucleotide is added to the growing polymer in a sequencing by synthesisreaction, the charge density at, or near the surface of the sensitivenanostructure may increase and this can be detected by a change inproperty in the sensitive detection nanostructure (for instance, ifusing a nanowire, or carbon nanotube, as the detecting structure, anincrease in charge caused by the addition of a nucleotide close to itssurface may be detected by a change in resistance in the wire, due to aphenomenon called the field effect). However, as the polymer grows, thesignal may diminish as the charges carried by the nucleotides beingadded may be too far away from the sensitive nanostructure (e.g.nanowire) to illicit a change in property of the sensitive detectionnanostructure and no signal may be observed. Therefore, the ‘readlength’ (amount of sequence data that is able to be obtained by thismethod of nucleotide sequencing) can be limited.

As used herein this particular example, a “sensitive detectionnanostructure” can be any structure (nanoscale or not) which can becapable of detecting any change in charge at, or near it's surface andat any point may have at least one cross-sectional dimension less thanabout 500 nanometers, typically less than about 200 nanometers, moretypically less than about 150 nanometers, still more typically less thanabout 100 nanometers, still more typically less than about 50nanometers, even more typically less than about 20 nanometers, stillmore typically less than about 10 nanometers, and even less than about 5nanometers. In other embodiments, at least on of the cross-sectionaldimensions can generally be less than about 2 nanometers, or about 1nanometer. In one set of embodiments the sensitive detectionnanostructure can have at least one cross-sectional dimension rangingfrom about 0.5 nanometers to about 200 nanometers.

The properties of a sensitive detection nanostructure may change inresponse to surface, or near surface charge in a way that may bemeasurable via piezoelectric measurements, electrochemical measurement,electromagnetic measurement, photodetection, mechanical, measurement,acoustic measurement, gravimetric measurement and the like. An exampleof a sensitive detection nanostructure may include, but not limited to,two dimension field effect transistors, a cantalevers, nanowires, carbonnanotubes, and all piezoelectric macro-, micro-, nano-, pico-, zempto-,or smaller structures.

Certain embodiments of the present disclosure may address thislimitation, at least in part by using synthetic nucleotides that maycomprise normal nucleotides, with a high negative (or positive) chargemass reporter moiety attached via a linker molecule (for instance,attached to the 5′ phosphate group), with the linker length increasingas the reaction progresses. This high charge mass can be designed to‘reach down’ to the sensitive nanostructure (e.g. nanowire) to cause achange in property of the sensitive detection nanostructure (e.g. afield effect or other piezo-electric change in the structure dependingon the sensitive detection nanostructure used). To enable a good qualitycontrol measure and to ensure long read lengths by eliminating the buildup of many reporter moieties which would cause an ever increasing fieldeffect, these reporter moieties can be cleaved at least in certainembodiments, to allow for the addition of the next nucleotide in thesequencing by synthesis sequence.

Therefore, in some embodiments the cyclical reaction may comprise atleast some or whole of the following entire or partial series of events:

-   -   1. The template ssDNA molecule to be sequenced can be either        ligated to the sensitive detection nanostructure and a primer        added, bind to a pre-immobilized primer sequence on the        sensitive detection nanostructure, or uncoiled and elongated in        a microfluidics channel arrayed with sensitive detection        nanostructures.    -   2. The sensitive detection nanostructures can be washed with        water, or a low salt buffer (such as 1×SSC). This washing,        however, may not be necessary in some embodiments.    -   3. A measure of the sensitive detection nanostructure can be        made.    -   4. A mixture containing one synthetic nucleotide, the polymerase        and other elements required for the polymerization reaction can        be added. In one example, if the nucleotide added is        complimentary to the base on the minus strand immediately after        the primer sequence, it maybe incorporated into the growing        chain by the polymerase.    -   5. The reaction can then be washed with either water or a low        salt buffer (such as 1×SSC). This washing, however, may not be        necessary in some embodiments.    -   6. A measure of the sensitive detection nanostructure can be        made which can observe the effect caused by the high charge mass        of the reporter moiety.    -   7. The reporter moiety can then be cleaved (for instance by an        acid solution or enzymatically). This cleavage of reporter        moiety, however, may not be necessary in some embodiments.    -   8. Points 2 through 7 can be repeated for each of the four        nucleotides. And this can be repeated repeatedly until a clear        signal may degrade.

For some embodiments wherein the template molecule is immobilized to, orbound to a probe that can be in turn immobilized to the sensitivedetection nanostructure, the linker lengths that attach the high chargereporter moiety to the synthetic nucleotides may increase to enable thecharge to ‘reach down’ to the sensitive detection nanostructure to exertan effect. This may be necessary at least in some embodiments as thegrowing nucleotide polymer may move the next nucleotide addition sitefarther and farther from the sensitive detection nanostructure as thesequencing by synthesis reaction may progress.

For some other embodiments wherein the template molecules is notimmobilized to the sensitive detection nanostructure, or hybridized to aprimer/probe that can be in turn immobilized to the sensitive detectionnanostructure, and can be instead free or immobilized horizontallyacross a cluster of sensitive detection nanostructures, a single linkerlength can be used for each of the cycle reactions

Example 2—Parallel Polony Sequencing

In one embodiment, genomic DNA may be fragmented by methods known tothose skilled in the art (such as sonication, restriction enzymedigestion, etc). Adaptamers (a short synthetic oligo nucleotide-DNA, orother synthetic oligonucleotide) can be then ligated to the fragments,an A adaptamer ligated to the 5′ end and a B adaptamer ligated to the 3′end.

The A adaptamer may contain a restriction enzyme binding site and the Badaptamer may be complimentary to another oligonucleotide immobilized toa nanostructure, such as a nanosphere. This oligonucleotide can be DNA,or any other synthetic nucleic acid and can generally be immobilizedwith methods familiar to those skilled in the art (for instance using astreptavidin coating on the nanostructure and biotin ligated to theterminal nucleotide in the oligonucleotide chain). These nanostructurescan generally be added to the library of small genomic fragments, withligated A & B adaptamers, in excess and then the whole mixture, with PCRreagents added, emulsified in oil, so that tiny microreactors ofnanostructure plus a single strand of the fragmented genome may allowfor huge multiplex amplification of the genome using the polymerasechain reaction. As the nanostructures can be added in excess, thedilution can be such that only one fragment may bind to anyonenanostructure. The amplified fragments, within each microreactor maynaturally bind to the nanostructures, as they are synthesised, which canbe coated in DNA (or another synthetic nucleotide, such as PNA,morpholinos, etc), complimentary to the B adaptamers. Thus eachmicroreactor, following the polymerase chain reaction, may comprise ananostructure coated in a single species of amplified genomic fragmentin certain parts of embodiments.

In some instances, these nanostructures, coated with a single species ofamplified genomic fragment can then be added to a special pico-titreplate, which may comprise hundreds of thousands of reaction wells. Atthe base of each well can be an array of individually addressedsensitive detection nanostructures coated with a ‘primer’, or ‘probe’oligonucleotide complimentary to the A adaptamer. The wells can be ofsuch a size to allow only one nanostructure in certain cases, coated inan amplified genomic fragment to rest in it. Once these nanostructurescan be allowed to rest in the pico-wells, the reaction plate can bewashed with a low salt buffer (such as 1×SSC).

In one embodiment, a measure of the sensitive detection nanostructureresistance can be taken as the baseline signal. Polymerase (the enzymethat can read the DNA and add complimentary bases) may be added, with asingle nucleotide (either Adenine Guanine, Cytosine or Thymine) andchemicals (such as MgCl₂) required for polymerase activity, can be addedand the sequencing by synthesis reaction can be performed, as describedabove, in parallel, in each well of the pico-titre plate.

In some embodiments, synthetic nucleotides, which have a charge moietyadded—as described above—can be used to amplify the signal. Furthermore,as the sequencing by synthesis reaction progresses and the nextnucleotide to be added to the nascent chain gets further from thesensitive detection nanostructure, the length of the linker moleculescan be increased to ‘reach-down’ to induce a signal in the sensitivedetection nanostructure from afar, thus enabling longer read lengths.

In other embodiments, following the wash step, a restriction digest canbe performed with the restriction enzyme cutting at the site in the Aadaptamer. This may release the nanostructure, which may be washed away.The sequencing by synthesis reaction can then be performed, as above,but without the presence of the nanostructure in the pico-well.

Example 3—Long Read-Length Sequencing in Microfluidics Channels or FlowCells

In some embodiments, target DNA sequences can be sequenced in amicrofluidics channel arrayed with sensitive detection nanostructures.

The genomic, or other nucleotide polymer molecule sample can befragmented into fragments > about 1 kb, but under about 10 kb and canhave an adaptamer (reverse compliment to the sequencing primer) ligatedto the 5′ end. These fragments can be then positioned within amicrofluidics channel, one per channel, either by hybridizing the 5′adaptamer sequence to an immobilized probe at the mouth of themicrofluidics channel and flowing a low salt buffer through the channelto draw the nucleotide polymer fragment into the channel, or by allowingthe nucleotide polymer to naturally diffuse and uncoil into themicrofluidics channel, or another method.

In such embodiment, once the nucleotide polymer fragment can be in themicrofluidics channel the sequencing by synthesis reaction can begin,with each arrayed sensitive nanostructure taking a measurement. Not onlymay these sensitive nanostructures detect the additions of bases, but asthe nascent chain may extend the spatial positions of each base additioncan be determined as each subsequent sensitive detection nanostructuremay begin to detect a signal, and those upstream may stop detecting asignal.

One of key aspects of such embodiments can be to ensure the surfacechemistry within the microfluidics channel prevents binding andaggregation of the charged reporter moieties or unincorporatednucleotides, as these may cause background noise.

Example 4—Handheld Sequencing Device

In some embodiment, specific target DNA sequences may be sequenced in amicrofluidics channel arrayed with sensitive detection nanostructures asabove. However, the sequencing by synthesis reagents can be stored in amicrofluidics channel, each nucleotide mix, wash solution and chargereporter moiety cleavage buffer, separated by an air bubble, or anothermethod of separating the reagents.

In some embodiment, small specific regions of target viral, bacterial orgenomic DNA can be to be sequenced to be diagnostic for the presence orabsence of a specific virus, bacteria, or sequence (such as a SNP).

Various embodiments used in connection with the present disclosure lenditself to handheld sequencing as it may not require bulky and expensivecameras or lasers to detect the sequencing reaction. Furthermore, byplacing the reagents one after the other in sequence, they may be easilymanipulated within a microfluidics environment.

The following figures are presented to provide some illustrative andnon-limiting examples of various embodiments,

FIG. 1 A. In one embodiment, a probe sequence can be immobilized on asensitive detection nanostructure (in this case a nanowire) and thetemplate ssDNA molecule to be sequenced can hybridize to the probesequence and the probe sequence can act as a primer for the sequencingby synthesis reaction. B. In this embodiment the template ssDNA moleculecan be immobilized to the sensitive detection nanostructure and can beprimed for sequencing with a free primer oligonucleotide.

In such embodiments, once the target DNA is bound to the immobilizedprimer/probe, or the primer hybridizes to the immobilized target DNA, ameasurement can be taken from the sensitive detection (for instance, ina nanowire or carbon nanotube, a resistance reading is taken).

FIG. 2 illustrates the next step in the sequencing by synthesis reactionin some embodiments, wherein a solution comprising at least some of asingle species of synthetic nucleotides, polymerase and chemicalrequired for polymerization can be added.

FIG. 3 illustrates the addition of a synthetic nucleotide base to thenascent chain in some embodiments. The ligated high negative charge massreporter moiety can extend from the synthetic nucleotide sequence and‘reaches-down’ to cause a measurable change in properties in thesensitive detection nanostructure (in this case a field effect in ananowire).

A second measurement of the sensitive detection nanostructure can betaken at this point.

FIG. 4 illustrates the linker and high charge mass reporter moietyligated to the added nucleotide can be cleaved, either enzymatically,with an acid, or other chemical or method, and washed away in someembodiments.

A third measurement of the sensitive detection nanostructure can betaken at this point.

The cycle of FIG. 2 through 4 can be repeated for the other threenucleotides and then again repetitively for all four until the desiredsequence can be obtained or the signal degrades.

FIG. 5 illustrates a depiction of the results from the measurements fromthe sensitive detection nanostructures after seven cycles consistingfollowing the following nucleotide additions Adenine, Cytosine, Thymine,Guanine, Adenine, Cytosine, and finally Thymine in some embodiments. Thefirst and second cycles that may cause a change in properties of thesensitive detection nanostructure, which can be interpreted as CT as thefirst two base pairs in the nascent strand. The third cycle that mayprovide a large signal, twice the order of magnitude that of the firsttwo cycles (this may be possible if the sensitive detectionnanostructure can be capable of quantitative or semi-quantitativemeasurements to differentiate between a dinucleotide stretch,tri-nucleotide stretch and other homopolymer stretches).

Alternatively, in some other embodiments, if the sensitive detectionnanostructure can be not able to differentiate between di-nucleotide,tri-nucleotide and other homopolymer additions, the syntheticnucleotides can be designed to allow the addition of, in one example,only one nucleotide and therefore may prevent the addition of othernucleotides. Upon cleavage of the linker and reporter moiety the abilityto add further nucleotides to the nascent chain can be restored.Therefore, at least in this example the sequence from this cycle wouldresult in ACTT only.

FIG. 6 illustrates a polymerase complex adding a synthetic nucleotidebase to the nascent chain as part of the polymerization reaction in someembodiments. Note the linker molecule protrudes out from the polymerasecomplex in this particular example, therefore not deleteriouslyaffecting its action, and ‘reaches-down’ to the sensitive detectionnanostructure (in this case a nanowire or carbon nanotube) to exert itseffect on the structure which can in turn be measured and recordedfollowing washing away the polymerase complex and chemicals required toenable it to work. Once the measurement can be taken the linker andreporter moiety can be removed and another measurement (baseline+1nucleotide) can be taken, before the next nucleotide in the sequencingby synthesis reaction may be added.

FIG. 7 illustrates a microfluidics cassette design designed for handheldsequencing in some embodiments.

a. Sample Reception—This element may act as a barrier for the sample toescape and can yet be able to accept samples, much like the rubber maytop on blood Vacutainers.

b. Lysis Chamber—This illustrates a simple microreactor, chamber whichcomprises a lysis reagent to break up the cells and to release genomicDNA. This section might also resemble a filter to remove blood cells ifthe target nucleotide polymer can be free in the blood serum.

c. Nucleic Acid Sample Preparation—The nucleotide polymer fraction ofthe sample can be isolated and extracted from the rest of the sampleconstituents (proteins, carbohydrates, lipids, etc). This can beachieved by some methods well known to those skilled in the art. Forinstance, this maco-fluidic chamber might contain Nexttec's filtertechnology.

d. Amplification of the Target Nucleotide Polymer—This section mayamplify the target nucleotide polymer, using the polymerase chainreaction, which may employ heating elements or other well knownstrategies of cycling a reaction mix through the different temperaturesrequired for PCR, to perform the thermal cycling required, or isothermalamplification methods (such as LAMB, RPA, etc), which may not requireheating of the sample.

e. Sample Processing—This might be required at least in some embodimentsto concentrate the nucleic acids, or remove ‘over-hang’ nucleotidechains that might cause background signal, prior to sequencing.

f. General Microfluidics—This describes the size of the channels, fluidflow, valves and control, materials and valves used in some embodiments.

g. Metal Connects—These connect the sensitive detection nanostructures(in this case, nanowires) to the detector device in some embodiments.

h. Sensitive Detection Nanostructure Arrays—The microfluidics channelcan be tightly arrayed sensitive detection nanostructures (such asnanowires, or carbon nanotubes). Two methods of positioning DNA in thechannel can be employed; 1. tight channels may allow long stretches ofDNA to uncoil, migrate & stretch down the channels which may allow forlong read lengths if necessary, and 2. tiling probe/primers can bespotted on to nanowire clusters and short multiple parallel sequencingreactions performed throughout the channels.

i. This weaving microfluidic channel can be filled with reagents in someembodiments, separated by air bubbles. As this microfluidics channel canbe pumped, or a tiny actuator moves the reagents along, the sequence ofthe reagents in the microfluidics channel can run the sequencing bysynthesis reaction.

What is claimed is:
 1. A method of sequencing a target polynucleotide, comprising: providing within an assay region a sensitive detection nanostructure that generates a signal related to a charge property within the assay region, wherein the sensitive detection nanostructure is operably coupled to a signal detector; hybridizing a primer to the target polynucleotide that is operably coupled to the sensitive detection nanostructure, such that the resulting primer-target polynucleotide is operably coupled to the sensitive detection nanostructure; adding one or more nucleotides and a polymerase to the primer-target polynucleotide within the assay region under conditions that support polymerization of a nascent chain when at least one of the added nucleotides is complementary to the base on the target polynucleotide downstream of the primer, wherein the added nucleotide comprises a charge mass reporter moiety comprising a high charge mass moiety and a linker; and detecting a change in the signal within the assay region upon addition of said nucleotide, wherein the change in the signal is characteristic of the at least one nucleotide added to the nascent chain and wherein the charge mass reporter moiety is removed from the added nucleotide after detecting the signal.
 2. The method of claim 1, wherein the charge property is sufficient to result in a detectable change in resistance of the nanostructure.
 3. The method of claim 1, wherein the charge mass reporter moiety is configured to be removable.
 4. The method of claim 1, wherein the charge mass reporter moiety is configured not to affect polymerization of the nascent chain by the polymerase.
 5. The method of claim 1, wherein the charge mass reporter moiety is configured to protrude out from the nascent chain so as to reach-down to the sensitive detection nanostructure.
 6. The method of claim 1, wherein the high charge mass moiety comprises an aromatic and/or aliphatic skeleton comprising one or more of a tertiary amino group, an alcohol hydroxyl group, a phenolic hydroxy group, and any combinations thereof.
 7. The method of claim 1, wherein the high charge mass moiety comprises one or more of the following groups or derivatives thereof:


8. The method of claim 1, wherein the linker comprises a molecule of the following general formula: H₂N-L-NH₂ wherein L comprises a linear or branched chain comprising an alkyl group, an oxy alkyl group, or a combination thereof.
 9. The method of claim 8, wherein L comprises a linear chain comprising an alkyl group, an oxy alkyl group, or a combination thereof.
 10. The method of claim 9, wherein a number of carbon atoms in the linear chain is 1 to
 100. 11. The method of claim 1, wherein the added nucleotide further comprises a cleavable cap molecule at the 5′ phosphate group so that addition of another nucleotide is prevented until the cleavable cap is removed.
 12. The method of claim 1, wherein the linker is bound to the 5′ phosphate group of the added nucleotide, thereby acting as a cap.
 13. The method of claim 1, wherein more than one nucleotides are added to the assay region, but wherein a successive nucleotide is not added to the nascent chain until after the signal that is characteristic of the preceding nucleotide added to the nascent chain is detected.
 14. The method of claim 1, wherein the operable coupling between the primer-target polynucleotide and the nanostructure comprises immobilization of the primer to the sensitive detection nanostructure.
 15. The method of claim 1, wherein the operable coupling between the primer-target polynucleotide and the nanostructure comprises immobilization of the target polynucleotide to the sensitive detection nanostructure.
 16. The method of claim 1, wherein hybridizing the primer to the target polynucleotide comprises hybridizing the primer to an oligonucleotide that has been ligated to the 5′ end of the target polynucleotide.
 17. The method of claim 1, wherein the sensitive detection nanostructure is selected from the group consisting of a nanowire, a nanotube, a nanogap, a nanobead, a nanopore, a field effect transistor (FET)-type biosensor, a planar field effect transistor, and any conducting nanostructures.
 18. The method of claim 1, wherein the target polynucleotide and the primer comprise molecules selected from the group consisting of DNA, RNA, peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), synthetic nucleotide polymer, and derivatives thereof.
 19. The method of claim 1, wherein the added nucleotide comprises a molecule selected from the group consisting of a deoxyribonucleotide, a ribonucleotide, a peptide nucleotide, a morpholino, a locked nucleotide, a glycol nucleotide, a threose nucleotide, a synthetic nucleotide, and derivatives thereof. 